Abstract Our recent clinical trial using adeno-associated viral vectors (AAV) to deliver Mini-Dystrophin to the muscle of patients with Duchenne muscular dystrophy (DMD) was met with an unexpected result; after treatment, a Dystrophin-specific T-cell response was found in two patients, which was related to revertant fiber development prior to therapy. These Dystrophin-specific T cells have the potential to eradicate all genetically modified muscle resulting in an ineffective therapy, as well as presenting a general concern for gene therapy communities in general. Currently, no strategy exists to avoid transgene-specific CTLs, whether they are pre-existing or therapy- induced, and systemic long-term immunosuppression is considered a non-viable option. However, particular viruses found in nature have evolved a potential solution to this dilemma by synthesizing small peptides that inhibit antigen presentation only in transduced cells. In preliminary experiments, we demonstrate that the cellular synthesis of these viral inhibitory peptides (termed VIPRs) prevents the surface presentation of a well defined antigen, thus protecting transduced cells from the host's immune response. In the current proposal, we will use a canine DMD model (GRMD) to evaluate the evasion ability of VIPRs from Dystrophin- specific CTL-mediated elimination of AAV transduced muscles. First, we will test whether the utilization of VIPRs can block the induction of a Dystrophin-specific CTL response after AAV muscle injection (Aim 1). Next, we will study whether the application of VIPRs will help AAV transduced muscle fibers escape pre-existing Dystrophin-specific CTL-mediated elimination (Aim 2). To decrease the vector cassette size for efficient virion package and/or to enhance the evasion ability of VIPRs, we will optimize the VIPR domains and test the immune evasion capacity of mutant variants (Aim 3). By delivering VIPRs and mini-dystrophin in the same vector, this approach ensures that antigen presentation will be attenuated only in AAV- transduced cells without systemic side effects on the immune system (as would be the case using immunosuppressive drugs or by the application of regulatory T-cells). Collectively, this proposal outlines a promising strategy to overcome our clinical DMD observations and concerns for gene therapy studies in general, by creating an AAV vector capable of avoiding the host's immune response to a foreign protein.